Form identification of purple flying squid (Sthenoteuthis

IntroductionPurpleback flying squid Sthenoteuthis oualaniensis is an abundant and highly productive species that inhabits open waters of the Indo-Pacific region, including tropical and subtropical waters (Jereb and Roper, 2010). This species is characterized by fast growth, short lifespan, and semelparous reproduction (Liu et al., 2016). S. oualaniensis plays a critical role in epipelagic to mesopelagic waters, preying on a wide spectrum of food organisms from mesozooplankton to myctophids and supporting diverse marine predators including squids, fishes, sharks, whales, and seabirds (Jereb and Roper, 2010). S. oualaniensis is also becoming an important fishery species (Zhang et al., 2014). In the central South China Sea (SCS), for example, S. oualaniensis is a major target for the small-scale jigging fishery (Chen et al., 2008) as well as for the large-scale light falling-net fishery (Yang, 2002; Zhang et al., 2013). Similar to other Ommastrephidae squid such as Dosidicus gigas and Illex argentinus (Keyl et al., 2011; Rodhouse et al., 2013), however, stock discrimination is still uncertain and an important research priority (Chen et al., 2012), particularly for the case of increasing fishing pressure in the SCS (Zhang et al., 2014).Stock structure information provides a basis for understanding the dynamics of cephalopod populations. Each stock may have unique demographic properties and responses to exploitation (Boyle and Rodhouse, 2005). Morphometric characteristics form the basis for one of the simplest, and most often used, tools to identify and characterize squid stocks, i.e., in determining population assemblages (Rodhouse et al., 2013) and assigning individuals to stock (Fang et al., 2014). Morphometric characteristics can also be used to identify traits with evolutionary significance (e.g., Wanninger and Wollesen, 2018). Therefore, information on how, and the extent to which, a species or one stock within a species evolves specific morphometric relationships can potentially contribute to better management and conservation (Clavel and Morlon, 2017), as well as leading to a better understanding of species evolution, ecology, and ultimately stock assessment (Laptikhovsky et al., 2017; Wright et al., 2018).S. oualaniensis exhibits a complicated intraspecific stock structure, with multiple morphological (Nesis, 1993), geographic (Chen et al., 2007; Yan et al., 2015), molecular (Staaf et al., 2010), and/or even spawning forms (Liu et al., 2008). In the SCS, available information indicates that there are probably two stocks of purpleback flying squid—the middle-sized (with dorsal photophore) and dwarf (without dorsal photophore) forms (Zhu et al., 2016; Wang et al., 2017; Li et al., 2019). These two stocks are, however, unconfirmed, presumably owing to a significant geographic overlap in morphology (Li et al., 2019). According to the different types of gladius, middle-sized individuals are divided into two forms: the middle-sized, typical form with double axes and the middle-sized not typical form with single axes (Jereb and Roper, 2010). Hard structures such as gladius, statoliths, and beaks are increasingly applied for stock discrimination of cephalopods (e.g., Liu et al., 2008; Fang et al., 2014; Fan et al., 2015) and show greater promise than the mantle and other body organs (Zhu et al., 2016; Wang et al., 2019). The gladius grows continuously throughout the lifetime of the species. The morphological characteristics and chemical composition of the gladius among species are obviously different, which is an important basis for the classification of cephalopods (Liu and Chen, 2010; Chen and Qiu, 2014). Little attention has been paid to morphological variations in gladius between different forms of the same species; it still needs relative information for classification (Gong et al., 2018).Here, we used “form” to refer to different stocks of S. oualaniensis, following Jereb and Roper (2010). We analyzed the morphometric characteristics of the gladius of the middle-sized form (with dorsal photophore) and dwarf without a dorsal photophore form of S. oualaniensis and used multivariate statistics to analyze the differences in morphometric characteristics of the gladius between the two forms, with the ultimate aim of acquiring novel insights into stock identification. Specifically, we examined the following questions: 1) how different are the gladius morphologic characteristics between forms and sexes of S. oualaniensis? and 2) can gladius morphologic characteristics be used to distinguish these forms and/or sexes? These results will help put forward our understanding of the structure of this species and also warrant consideration of using the work as a framework to be applied in other commercially exploited squid species.Material and methodsSample collectionS. oualaniensis was sampled at 20 stations in the SCS (9.80°N–17.25°N, 110.25°E–115.02°E) by the Chinese lighting falling-net vessel Gui Beiyu 61999, from May to June in 2017 and 2018 (Figure 1). Four hundred thirty-eight specimens were randomly caught and collected and immediately frozen (–18°C) on board for further laboratory analysis.Figure 1 Study region (A) and stations (B) for Sthenoteuthis oualaniensis sampled in the South China Sea.After being defrosted at room temperature in the laboratory, S. oualaniensis was categorized into the dwarf without a dorsal photophore and middle-sized forms based on the absence or presence of the dorsal photophore and the apparatus’ length (Zhu et al., 2016). The middle-sized forms were categorized into the typical and not typical forms based on the double axes or single axis of the gladius (Jereb and Roper, 2010). The dorsal mantle lengths (ML) were measured to the nearest 1 mm and body mass (BW) weighted to the nearest 1 g. Thereafter, a subsample of 275 specimens (130 females, 145 males) were used for gladius morphology analysis, whereas the gladius of other samples had been abandoned due to damage. The subsampled S. oualaniensis were dissected, sexed, and assigned a maturity stage on a macro scale following Lin (2015): I immature, II developing, III physiologically maturing, IV–V physiologically mature, VI functionally mature, VII spawning, and VIII spent.Gladius morphometric measurementThe gladii of each subsampled S. oualaniensis were removed and measurements made of the following morphometric characteristics (Figure 2): total gladius length (GL), cone length (CL), greatest width of the cone (GWC), proostracum length (PL), length of the vane (VL), greatest width of the vane (GWV), the length of greatest width of the proostracum (GWPL), and greatest width of the proostracum (GWP). All these characteristics were measured accurately to be 0.01 mm.Figure 2 Scheme of gladius morphometric measurements for Sthenoteuthis oualaniensis. GL, total gladius length; CL, length of the cone; GWC, greatest width of the cone; PL, proostracum length; VL, length of the vane; GWV, greatest width of the vane; GWPL, length of the greatest width of the proostracum; GWP, greatest width of the proostracum.Statistical analysisEach morphometric measurement of the gladius was checked for normal distribution with the one-sample Kolmogorov–Smirnoff test as well as for homogeneity of the variances with the Levene’s test (Zar, 1999). One-way ANOVA was used to test the difference between different forms. When significant differences were achieved, a Tukey’s post-hoc test was applied to determine where the differences occurred (Zar, 1999). When either of normal distribution or homoscedasticity was not achieved, data were subjected to a Kruskal–Wallis non-parametric one-way ANOVA test and a Games–Howell post-hoc test was performed (Zar, 1999).Non-metric multidimensional scaling (nMDS) analysis and analysis of similarity (ANOSIM) employing the Bray–Curtis dissimilarity measure were used to access the dissimilarities of gladii between forms. This allowed for the potential identification of individuals that belong to the dwarf form without a dorsal photophore or middle-sized form. Furthermore, a stepwise discriminant analysis was performed to identify the gladius morphological characteristics that significantly classified individuals from the dwarf without a dorsal photophore and middle-sized forms (Rencher, 2002). The leave-one-out cross-validation was used to determine the rate of correct classification for different groups.
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